A neutral CB1 receptor antagonist reduces weight gain in rat

A neutral CB1 receptor antagonist reduces weight gain in rat.

Adam P. Chambers1, V. Kiran Vemuri2, Yan Peng2, JodiAnne T. Wood2, Teresa

Olszewska2, Quentin J. Pittman1, Alexandros Makriyannis2, Keith A. Sharkey1.

1Hotchkiss Brain Institute and Institute of Infection, Immunity and Inflammation,

Department of Physiology & Biophysics, University of Calgary, AB, Canada;

2Center for Drug Discovery, Northeastern University, Boston, MA, USA.

These studies were supported by grants from Canadian Institutes of Health

Research (to QJP and KAS) and from National Institutes of Health, U.S.A

(DA09158, DA7215, DA3801 to AM). APC is an Alberta Heritage Foundation for

Medical Research (AHFMR) Graduate Student. QJP and KAS are AHFMR Medical

Scientists. KAS is the Crohn’s and Colitis Foundation of Canada Chair in

Inflammatory Bowel Disease Research and QJP holds a University Professorship.

*Correspondence should be addressed to these authors at:

Keith A. Sharkey, PhD, Department of Physiology & Biophysics, University of

Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada; Tel.: 1-403-220-

4601; Fax: 1-403-283-3028; Email: [email protected]

Alexandros Makriyannis, PhD, Center for Drug Discovery, 116 Mugar Hall,

Northeastern University, Boston, MA, 02115 USA; Tel: 1-617-373-4200; Fax: 1-

617-373-8886; Email: [email protected]

Running Head: AM4113 Reduces Weight Gain In Rat

Page 1 of 40Articles in PresS. Am J Physiol Regul Integr Comp Physiol (October 24, 2007). doi:10.1152/ajpregu.00663.2007

Copyright © 2007 by the American Physiological Society.

AM4113 Reduces Weight Gain In Rat 2

ABSTRACT

Cannabinoid (CB) 1 receptor inverse agonists inhibit food intake in animals and

humans, but also potentiate emesis. It is not clear whether these effects result

from inverse agonist properties or from the blockade of endogenous cannabinoid

signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on

food intake, weight gain and emesis. Neutral antagonist and binding properties

were confirmed in HEK293 cells transfected with human CB1 or CB2 receptors.

AM4113 had no effect on forskolin stimulated cAMP production at concentrations

up to 630 nM. The Ki value of AM4113 (0.80±0.44nM) in competitive binding

assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1

over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in

brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg kg-1)

significantly reduced food intake and weight gain in rat. Compared with AM251,

higher doses of AM4113 were needed to produce similar effects on food intake

and body weight. Unlike AM251 (5 mg kg-1), a highly anorectic dose of AM4113

(10 mg kg-1) did not significantly potentiate vomiting induced by the emetic

morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor

antagonist shares the appetite suppressant and weight loss effects of inverse

agonists. If these compounds display similar properties in humans they could be

developed into a new class of anti-obesity agents.

Keywords: cannabinoid receptor, food intake, inverse agonist, obesity, emesis,

hypothermia.

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INTRODUCTION

Cannabinoid (CB)1 G-protein coupled receptors, along with endogenous

cannabinoids (endocannabinoids), including anandamide and 2-

arachidonylglycerol (2-AG) and the biosynthetic and degradative enzymes for

these ligands are found in pathways involved in feeding and metabolism (33).

These pathways include vagal afferents (5), area postrema and brainstem nuclei

(40; 56), hypothalamus (11; 52), and reward pathways (29; 50). In the periphery,

CB1 receptors and their ligands are also found in the gut (20), liver (43), and

adipose tissue (2; 11; 28). Vagal afferents, area postrema and brainstem are

also involved in emesis (26; 55; 56).

Therapeutically, CB1 receptor agonists are used as anti-emetics and to

promote appetite during serious illness such as cancer (14) and acquired

immune deficiency syndrome (AIDS) (58). In the case of cancer, nausea and

emesis induced by chemo- and radiation therapy is significantly attenuated by the

active ingredient in marijuana, delta-9-tetrahydrocannbinol, and other CB1

receptor agonists (14; 58). CB1 receptor inverse agonists such as SR141716A

and the structurally, and pharmacologically similar compound AM251 are also

therapeutically relevant. These agents suppress appetite, promote weight loss,

and improve cardiometabolic risk factors in humans (17; 46; 54) and rodents (7;

19; 22; 23; 25; 28; 47; 53; 57). However, significant psychological and

physiological concerns in patients treated with CB1 receptor inverse agonists are

also present including increased risks of nausea and vomiting (17; 46; 54).

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Previously, CB1 receptor inverse agonists were shown to promote emesis in

ferret (56) and shrew (13), and promote nausea (37) in rat.

At CB1 receptors, agonist activity decreases adenylyl cyclase and the

conversion of adenosine triphosphate (ATP) to cyclic adenosine 3’,5’-

monophosphate (cAMP) (18; 45). Like an antagonist, inverse agonists block

receptor binding and activation by a competitive agonist, but in addition, they also

inhibit intrinsic or spontaneous receptor signaling (42). The result is an effect

opposite to that produced by a given agonist and is termed inverse agonism.

Although no valid method for assessing inverse agonist activity in vivo exists

(42), the inhibition of intrinsic CB1 receptor transduction can be shown in vitro by

the increased expression of adenylyl cyclase and cAMP in cultured cells

transfected with CB1 receptors (35; 39; 51). Whether the physiological and

behavioral effects of CB1 receptor inverse agonists result from the ability of these

compounds to inhibit intrinsic receptor activity, or, from the pharmacological

blockade of endogenous cannabinoid signaling, or, both, is not known. However,

a recent study by Sink et al, 2007 (51) describing a novel CB1 receptor

antagonist, AM4113, with no inverse agonist properties shows that such

determinations are now possible. These authors demonstrated that AM4113

reduced food intake in rat with no effect on conditioned gaping, a specific marker

of nausea in that species (51), suggesting that pharmacological blockade of CB1

receptors alone is sufficient to reduce food intake independent of nausea.

However, in that study food intake was only examined during a 30 min

observation period (51). The effect of a neutral CB1 receptor antagonist on daily

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food intake and body weight is not known. Given that in some cases (2; 10; 23;

28; 47; 49), but not others (7; 53; 57), weight loss induced by CB1 receptor

inverse agonists is unrelated to their effect on feeding, studies that examine the

effect of neutral CB1 receptor antagonism on body weight are crucial. In addition,

rats lack the neuronal pathways required for vomiting, and the effect of a neutral

CB1 receptor on emesis, which is distinct from nausea, is also unknown. In the

present study, we confirm the neutral antagonist and binding properties of

AM4113. We then test the ability of AM4113 to antagonize hypothermia induced

by the non-selective cannabinoid CP55,940 in rat. We chose this assay because

the mild hypothermia caused by cannabinoid agonists is mediated by CB1

receptors in brain (4; 21), enabling us to determine if AM4113 crossed the blood

brain barrier. We also examine the effect of AM4113 on food intake and body

weight in rats treated daily for 5 d and compare the acute effect of the antagonist

on food intake with the inverse agonist AM251 directly. Finally, we examine the

effect of AM4113 on emesis in ferrets treated with the emetic morphine-6-

glucoronide (M6G) using vehicle and AM251 treated ferrets for comparison.

MATERIALS & METHODS

Forskolin-Stimulated cAMP Assay

Intracellular cAMP levels were measured with a competitive protein-

binding assay using intact HEK293 cells expressing hCB1 or hCB2 receptors, and

a cAMP immunoassay kit from Sigma (St. Louis, MO, USA). In short, forskolin

stimulated cells were incubated with various concentrations of AM4113 or

Page 5 of 40


AM251, cAMP antibody and cAMP conjugate for 2 hours at ambient temperature.

The reaction was stopped by emptying the wells followed by the addition of p-

NPP substrate and incubation for 1 hour. Absorbance intensity, detected at 405

nm, was inversely proportional to the concentration of cAMP produced by the

cells. The results are expressed as the percent stimulation of forskolin-

stimulated cAMP accumulation.

[3H]CP55,940 Competitive Binding Assay

Compounds were tested for their CB1 and CB2 receptor affinity using

membrane preparations from rat brain (CB1 receptors) or HEK293 cells

expressing human CB2 (hCB2) receptor, respectively, and [3H]CP55,940 as

previously described (30; 32; 35; 41). Stock solutions of the compounds [10 mM

in dimethyl sulfoxide (DMSO)] were diluted in TME buffer (50 mM Tris-HCL, 3

mM MgCl2, 100 mM NaCl, 0.2 mM EDTA, pH 7.4) with 0.1% BSA and

transferred to 96 well plates containing [3H]CP55,940 (specific activity 128

Ci/mmol; NIDA) at a final concentration of 0.76 nM. Non-specific binding was

assessed in the presence of 100 nM CP55,940. The binding reaction was

initiated with the addition of the respective membrane suspension (~50 µg

membrane protein) followed by incubation at 30°C with gentle agitation in a

shaking water bath for 60 minutes. Binding was terminated by rapid filtration of

the membrane suspension over Unifilter GF/B-96 Well Filter Plates (Packard

Instruments) using a Packard Filtermate-196 Cell Harvester. The filter plates

were washed with ice-cold wash buffer (50 mM Tris-base, 5 mM MgCl2 with 0.5%

Page 6 of 40


BSA) and bound radioactivity was determined using a Packard TopCount

Scintillation Counter. All data were in duplicate with IC50 and Ki values

determined from at least two independent experiments.

Behavioral Experiments

All experimental protocols were approved by the University of Calgary Animal

Care Committee, and were carried out in accordance with the guidelines of the

Canadian Council on Animal Care. Different concentrations of drugs were

administered i.p. in a 1 ml kg-1 volume, except M6G, which was given s.c. in a 0.1

ml kg-1 volume.

Antagonism by AM4113 at CB1 receptors in brain

We examined hypothermia induced by the CB1 agonist CP55,940 (0.3 mg

kg-1) (15) in male Sprague Dawley rats (Charles River, Montreal, Qc, Canada)

pre-treated 45 min earlier with either vehicle, AM251 (5 mg kg-1, n=5) (7), or

AM4113 (5 mg kg-1, n=5). Briefly, silicone coated temperature data loggers

(SubCue Inc, Calgary, Alta, Canada) were surgically implanted into the

abdominal cavity of rats (450-500g) under isoflurane anesthesia (4% induction; 2-

2.5% maintenance). Rats recovered for 3 days before being acclimatized to

testing and handling procedures for an additional seven days. During the

experiment core body temperature readings were sampled every 5 min for 300

min. Apart from the injection procedure the rats were not handled during the

experiment.

Page 7 of 40


Food Intake Studies

Male Sprague-Dawley rats weighing between 330-380 g at the start of the

study were used to examine the effect of AM4113 on food intake and body

weight. Animals were fed strawberry flavored Ensure Plus® liquid diet (53.3%

carbohydrate, 29% fat, and 16.7% protein; 1.41 kcal g-1) (Abbott Laboratories,

Abbott Park, IL, USA) to promote food intake (1) and control for spillage. Rats

were habituated to testing and handling procedures, daily, for seven days prior to

testing. Food and water were presented in drip-free inverted glass bottles

attached to the outside of the cage. Food was available for 18 h each day starting

at 16:00 h (12 h light-dark cycle; lights off 16:00 h). Bottles were removed and

washed at 09:00 h, a time during the light-dark cycle when rats are generally

inactive.

Experiment 1

In experiment 1 we examined the effect of AM4113 (1 mg kg-1; 5 mg kg-1)

in rats treated daily for 5 days with the neutral antagonist. Doses were chosen

based on our thermoregulation data and previous work by our group with the

inverse agonist AM251 (7; 8; 36; 37). The day before the experiment rats were

assigned to one of three treatment groups: vehicle [mean bodyweight ± standard

error of the mean (SEM); (363±10 g, n=5)], AM4113 1 mg kg-1 (365±5 g, n=5),

AM4113 5 mg kg-1 (361±9 g, n=5). Food intake and body weight were monitored

daily for 5 d.

Experiment 2

Page 8 of 40


In experiment 2 the effect of AM4113 (10; 20 mg kg-1) on food intake and

body weight were compared to rats treated with vehicle or the inverse agonist

AM251 (5 mg kg-1). On the day before the experiment rats were randomly

assigned to one of four treatment groups: vehicle (492±8 g, n=6), AM251 5 mg

kg-1 (496±11 g, n=6), AM4113 10 mg kg-1 (491±7 g, n=6), or AM4113 20 mg kg-1

(489±9 g, n=6). Rats were injected between 15:00 and 16:00 h on the day of the

experiment. Treatments were counterbalanced to avoid order effects. After

treatment food was made available as described in experiment 1. Food intake

was measured 1.5, 3, 5, and 18 h after treatment. After the experiment food

intake and body weight were monitored for another five days to assess the

sustained effect of each compound after a single treatment.

Emesis Studies

We examined how AM4113 (10 mg kg; n=5) affected vomiting in ferrets

(Marshall Farms, North Rose, NY, USA) using vehicle (n=6) and AM251 (5 mg

kg-1) treated ferrets for comparison. Each treatment was given 15 min prior to

receiving the emetic M6G (0.05 mg kg-1) (56) . Ferrets were lightly anaesthetized

with halothane for each injection. Data were videotaped and analyzed by an

observer blinded to the conditions of each treatment group. During 60 min of

observation the number of vomiting (emetic) episodes were counted, and activity

or sleeping time was noted. In a second experiment the effect of two additional

doses of AM4113 (5; 20 mg kg-1; n=5/5) on M6G induced emesis were

examined. An additional group of vehicle (n=6) treated ferrets were used for

Page 9 of 40


comparison. Experiments were carried out using a counterbalance design to

avoid order effects.

Statistics

EC50 curves were generated from the forskolin-stimulated cAMP assay

data by non-linear regression with the use of GraphPad Prism software

(GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego

California USA). Data are expressed as mean % change in forskolin stimulated

cAMP accumulation ± SEM. Results from the competitive binding assay were

analyzed using nonlinear regression to determine the actual IC50 and the Ki

values of the ligand (GraphPad Prism) (9). Data are expressed as mean Ki in nM

± standard deviation. Food intake and thermoregulation data were analyzed

using a 2-way mixed design ANOVA with time as the repeated measure; Food

intake data are expressed as mean food intake in kilocalories (kcal) ± SEM. Body

temperature data are expressed in °C ± SEM. Significant differences were

followed up with 1-way independent measures ANOVA at each time point.

Significant differences between treatments were further analyzed using Newman-

Keuls multiple comparisons test. The emesis data comparisons between vehicle,

AM4113 (10 mg kg-1) and AM251 (5 mg kg-1) were made using a one-way

analysis of variance. Significant differences were followed up using Newman-

Keuls multiple comparisons test. Data examining the effect of additional doses of

AM4113 (0-20 mg kg-1) on emesis were analyzed using a one-way analysis of

Page 10 of 40


variance, and then by linear regression analysis. Emesis data are expressed as

mean number of vomiting episodes ± SEM.

Compounds

AM4113; N-piperidin-1-yl-2,4-dichlorophenyl-1H-pyrazole-3-carboxamide

analog (partial designation). AM4113 is a pyrazole congener of AM251 with a

very similar molecular weight. AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-

dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide. AM251 and AM4113 were

synthesized at Northeastern University. CP55,940 (-)-cis-3-[2-Hydroxy-4-(1,1-

dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; Tocris Cookson

Inc., Ellisville, MO, USA or NIDA, USA), and morphine-6-glucoronide (M6G,

Lipomed Inc., Arlesheim, BL, Switzerland). All compounds were dissolved in

DMSO using gentle heating and sonication before being diluted with Tween 80

and saline (4% DMSO; 1% Tween 80; 95% saline), and given in a volume of 1 ml

kg-1. Differences between baseline and vehicle (4% DMSO; 1% Tween 80; 95%

saline) conditions were non-significant at all times.

RESULTS

Forskolin-Stimulated cAMP Assay

Consistent with results reported by others (51) AM4113 did not change the

forskolin-stimulated cAMP accumulation in CB1 transfected HEK cells at

concentrations up to 630 nM and is therefore considered to be a CB1 neutral

antagonist (data not shown). In comparison, the inverse agonist AM251

Page 11 of 40


increased forskolin stimulated cAMP production in a concentration dependent

manner (EC50 56.4 nM; 95% confidence interval 5.5-573.8 nM, R2=0.49). The

results are from one assay performed in duplicate.

[3H]CP55,940 Competitive Binding Assay

In competitive receptor binding assays against [3H]CP55,940 AM4113

showed selectivity for the CB1 receptor compared to CB2. AM4113 bound to

cannabinoid receptors with a CB1 Ki of 0.80 nM and a CB2 Ki of 97 nM, indicating

that AM4113 exhibits at least a 100-fold selectivity for CB1 versus CB2 receptors

consistent with data by others (51). In comparison, AM251 bound to cannabinoid

receptors with a CB1 Ki of 3.43 nM and a CB2 Ki of 124 nM (data not shown).

AM4113 in Brain: Thermoregulation Assay

To determine whether AM4113 antagonized CB1 receptors in brain we

examined hypothermia induced by CP55,940 in rats pretreated with either

vehicle, AM251 (5 mg kg-1), or AM4113 (5 mg kg-1). Figure 1a shows changes in

body temperature over time. A 2-Way ANOVA performed on the body

temperature data showed a significant treatment by time interaction, F=4.4,

P<0.001. Differences between treatments were analyzed using a 1-way ANOVA

at selected time points (Figure 1b). Prior to treatment with CP55,940, differences

in body temperature between groups were non significant, P>0.05, indicating

that AM4113 and AM251 alone had no effect on core body temperature. As

expected, body temperature rapidly fell in vehicle treated rats after the

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administration of CP55,940 (15). In comparison, the hypothermic effect of

CP55,940 was completely blocked in rats treated with the neutral antagonist

AM4113, P<0.05, and significantly attenuated in rats pre-treated with inverse

agonist AM251, P<0.05.

Food Intake & Body Weight Studies

In experiment 1 we examined how daily treatment with AM4113 (1, 5, mg

kg-1) affected food intake over 5 days (Figure 2a). A 2-way ANOVA performed on

the food intake data showed that there was a significant treatment by time

interaction, F= 3.3, P<0.001. There was also a significant effect of dose, F=3.6,

P< 0.001. 1-way ANOVA performed between treatments showed that food intake

was significantly reduced in rats treated with 5 mg kg-1 of AM4113 on days 3, 4,

and 5 compared with vehicle and 1 mg kg-1 treated rats, P<.05, Newman-Keuls

Multiple Comparison Test.

Reductions in food intake by AM4113 (5 mg kg-1) were accompanied by

significant reductions in weight gain (Figure 2b). A 2-way ANOVA revealed a

significant effect of dose F=15.2, P<0.001, and, time F=15.4, P<0.001. Rats

treated with 5 mg kg-1 of AM4113 gained significantly less weight than rats

treated with 1 mg kg-1 of the neutral antagonist on day 4 (-16.9 kcal), P<0.05, and

significantly less weight than vehicle treated rats on day 4 (-22.0 kcal), day 5 (-

30.7 kcal), and day 6 (-33.4 kcal). Although rats treated daily with the 1 mg kg-1

dose of AM4113 tended to eat less, and gain less weight, than vehicle treated

rats such differences were not significant, P>0.05. The results demonstrate that

Page 13 of 40


repeated administrations of a 5 mg kg-1 dose of the neutral antagonist AM4113

significantly reduced food intake and weight gain in rat.

In experiment 2, we examined the acute effect of AM4113 on food intake

by evaluating two additional doses of the neutral antagonist (10; 20 mg kg-1)

compared with vehicle treated rats, and rats treated with the inverse agonist

AM251 (5 mg kg-1). Figure 3a shows the early effect of AM4113 and AM251 on

food intake. Results from a 2-way ANOVA performed on the data revealed a

significant treatment by time interaction, F=18.1, P<0.001. At each time point

food intake was reduced in rats treated with AM4113 (10; 20 mg kg-1) compared

to vehicle treated rats; 1.5 h (-20.2 kcal, -21.2 kcal), 3 h (-28.2 kcal, -30.0 kcal), 5

h (-32.1 kcal, -42.4 kcal), and 18 h (-59.2 kcal, -85.4 kcal), P<0.05. As expected

(8), AM251 produced a similar effect on feeding, P<0.05. Differences in food

intake between 10 and 20 mg kg-1 treatment groups were significant 18 h after

treatment, P<0.001. Differences between rats treated with AM251 and AM4113

were significant at 3, 5, and 18 h time points, P<0.05. The results demonstrate

that the neutral receptor antagonist AM4113 produced early, and dose-

dependent, reductions in food intake in rat.

Furthermore, continued monitoring revealed that the anorectic effect of the

neutral antagonist AM4113 (10; 20 mg kg-1) was sustained for several days after

treatment. Figure 3b shows the mean daily food intake in each group before and

after the experiment. A 2-way ANOVA performed on the daily food intake data

revealed a significant treatment by time interaction, F=12.7, P<0.001. Prior to

treatment, differences in food intake between groups were non-significant,

Page 14 of 40


P>0.05. After treatment, food intake was reduced in rats given AM251 or

AM4113 relative to vehicle treated rats, P<0.05. The sustained effect of an acute

administration of AM251 has been reported previously by our group (7; 8). Here

we report that reductions in food intake by the neutral antagonist AM4113 were

similar in duration to the anorectic effect of AM251.

Figure 3c shows that reductions in food intake by AM251 and the neutral

antagonist AM4113 (10; 20 mg kg-1) were also associated with significant

reductions in weight gain. Results from a 2-way ANOVA performed on the weight

change data showed that there was a significant treatment by time interaction,

F=10.9, P<0.001. Weight gain was significantly reduced in rats treated with

AM4113 or AM251 for several days after the experiment; P<0.05, Newman-Keuls

Multiple Comparisons Test. A 2-way ANOVA performed on the weight change

data also showed a significant effect of dose, F=27.9, P<0.001. Differences in

weight gain between 10 and 20 mg kg-1 AM4113 treatment groups were

significant on day 5 (-12.3 g), P<0.05. The results demonstrate that a single

administration of the neutral antagonist AM4113 (10;20 mg kg-1) produced a

dose-dependent reduction in weight gain and food intake.

Emesis Studies

We examined how AM4113 (10 mg kg; n=5) affected emesis in ferrets

compared with vehicle (n=6) treated ferrets, or, ferrets treated with the inverse

agonist AM251 (5 mg kg-1). Each ferret was given M6G 15 min after treatment to

induce emesis (Figure 4a). Doses were chosen based on work from our feeding

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studies showing that a 10 mg kg-1 dose of AM4113 produced roughly the same

effect on food intake as a 5 mg kg-1 dose of AM251 (Figure 3). A 1-way ANOVA

performed on the data showed that there was a significant effect of treatment,

F=4.1, P<0.05, caused by increased vomiting in AM251 treated ferrets compared

to vehicle treatment, P<0.05, Newman-Keuls Multiple comparison test. No

significant difference in vomiting episodes between AM4113 and vehicle treated

ferrets was observed. However, there was also no significant difference between

ferrets treated with AM4113 and AM251, P>0.05. In order to test the effect of

AM4113 on emesis more rigorously, two additional doses of AM4113 (0;5;20 mg

kg-1) were examined and compared to a new group of vehicle treated ferrets

(Figure 4b). A 1-way ANOVA performed on the data revealed no significant effect

of treatment, P>0.05. However, when the data was analyzed using linear

regression analysis a significant relationship between the dose of AM4113 and

number of vomiting episodes was shown, r2=0.96, P=0.04. The results suggest

that although AM4113 is less pro-emetic than the inverse agonist, AM251,

pharmacological blockade of endogenous cannabinoid signaling is capable of

affecting emesis in ferret.

DISCUSSION

We compared the effects of a novel CB1 receptor neutral antagonist,

AM4113, on food intake and body weight with those of the well characterized

inverse agonist AM251. Neutral antagonist properties were established in

HEK293 cells transfected with human CB1 receptors. We confirmed that AM4113

Page 16 of 40


acted as a neutral antagonist in vitro in HEK cells transfected with human CB1

receptors. Consistent with results by others (51), AM4113 had no effect on cAMP

at concentrations up to 630 nM and displayed low Ki values in competitive

binding assays with the CB1 agonist [3H]CP55,940. In vivo, AM4113 antagonized

the hypothermic effect of the CB1 receptor agonist CP55,940 and significantly

reduced food intake and weight gain in rat. Compared with AM251, higher doses

of the neutral antagonist AM4113 were needed to produce similar effects on food

intake and weight gain. Unlike AM251, AM4113 did not significantly increase

vomiting induced by the emetic M6G, however, there was a positive correlation

between dose and vomiting in ferrets treated with the neutral antagonist.

The fact that AM4113 blocked the hypothermic effect of a CB1 agonist

demonstrates that the neutral antagonist crosses the blood-brain barrier,

because the hypothermic effect of cannabinoid agonists is specific to activity at

CB1 receptors in the anterior hypothalamus (4; 21). Consistent with these

observations, preliminary studies performed on mice in our lab show that 0.4-

0.7% (Initial Dose/g brain) of an intravenously injected 2 mg/kg dose is found in

brain 15 minutes post-injection (data not shown). Together with a high brain

(µg/g) to plasma (µg/mL) ratio (0.49 ± 0.029), these data show that AM4113

crosses the blood-brain-barrier to affect CB1 receptors in the CNS. Moreover,

both AM4113 and AM251, alone, had no effect on core body temperature in the

45 min prior to treatment with the CB1 agonist CP55,940. These data

demonstrates that endogenous cannabinoid tone is not involved in

thermoregulation under the conditions we described, even with the inhibition of

Page 17 of 40


intrinsic CB1 receptor activity by AM251. Similar effects on temperature with CB1

receptor inverse agonists were shown previously (15; 38).

In contrast to our thermoregulation assay, larger doses of the neutral

antagonist AM4113 were needed to elicit the same effect on food intake as

AM251. AM4113 only affected daily food intake and body weight in rat at a 5 mg

kg-1 dose after several treatments. Whereas the same dose of the inverse

agonist, AM251, produced an immediate and sustained anorectic effect that

lasted for several days. If reductions in food intake by these agents were

produced solely from pharmacological blockade at CB1 receptors alone, then

matching doses of each compound should produce similar effects on feeding.

This was not the case in our study. Instead, our data suggest that the inverse

agonist properties of AM251 may contribute to the ability of this compound to

affect feeding pathways either directly or, possibly indirectly through feelings of

nausea and malaise (37; 51). However, we previously showed that rats do not

avoid flavored food pellets associated with the effects of AM251 at this dose (7)

even when alternative flavors paired with vehicle treatment are available

(conditioned taste aversion). However, the fact that AM251 increases

conditioned gaping (nausea) in rat (37; 51), combined with higher instances of

nausea in humans treated with the CB1 receptor inverse agonist SR141716A (17;

46; 54), shows that we cannot completely rule this out as a possibility. Both

AM251 (37) and the inverse agonist SR141716A (16) are capable of producing

conditioned taste aversion. Regardless, the fact that a neutral antagonist is

capable of producing reductions in food intake and body weight, independent of

Page 18 of 40


nausea and emesis, demonstrates that energy balance is controlled, in part, by

the release and action of endogenous cannabinoids. Because of the inverse

agonist properties of AM251, it has been previously impossible to draw this

conclusion. We note that the effect of another potential CB1 antagonist, LH-21,

on food intake and body weight has recently been reported (44), but that the

neutral antagonist properties of this compound have yet to be established (27).

The sustained effect of AM251 on food intake reported previously (7; 8)

and in the present study may result from the long half-life (~22 h) of this

compound in rat (36). One explanation for the delayed onset of the anorectic

effect of AM4113 in our 5 d feeding study is that sufficient quantities of the

compound needed to accumulate in order to become effective. If reductions in

food intake by AM4113 resulted from the build up of the neutral antagonist over

successive treatments, then rats treated acutely with higher doses of the

compound should show an immediate and sustained reduction in feeding.

Consistent with this hypothesis, results from experiment 2 show that higher

doses of AM4113 produced an immediate reduction in food intake, and, later

weight gain that persisted for several days after treatment. In our study, the time

course with which AM4113 reduced food intake and body weight was similar to

that seen with the inverse agonist AM251. Currently, there is no data available

regarding the half-life of AM4113.

CB1 receptor inverse agonists may cause nausea and are proemetic in

humans (17; 46; 54) and animals (13; 37; 56). We induced emesis in ferrets pre-

treated with the neutral antagonist AM4113 and found that there was a positive

Page 19 of 40


correlation between the dose and the number of vomiting episodes produced by

the emetic. These and other data (26; 55; 56) show that emesis likely stimulates

the release of endogenous cannabinoids which in turn serve to inhibit emesis in a

negative feedback manner. We also show that the inverse agonist properties of

AM251 likely contribute to its effect on emesis. AM4113 antagonized the

hypothermic effect of CP55,940 as effectively as AM251 in rat, but was

substantially less proemetic than the inverse agonist, in so far as AM4113 did not

significantly increase the mean number of vomiting episodes relative to vehicle

treatment. These data suggest that the inverse agonist properties of AM251 do in

fact contribute to its effect on emesis, and is consistent with results by Sink et al,

2007 showing a similar effect on nausea in rat. In this way, a neutral antagonist

such AM4113 may be better tolerated than inverse agonists as antiobesity

agents.

The CB1 receptor inverse agonist SR141716A (Acomplia/Rimonabant™)

has already been shown to significantly improve central obesity, cholesterol

profiles, circulating triglyceride levels and insulin resistance in overweight and

obese humans (17; 46; 54). In humans, circulating levels of the endogenous

cannabinoid 2-AG are positively correlated with intra-abdominal obesity, (3; 12),

and CB1 receptor message is downregulated in visceral adipose tissue of obese

subjects (3). These and other studies (6; 22-25; 28; 29; 31; 34; 43; 46; 48; 53; 54;

57) support the view that changes in endogenous cannabinoid signaling may in

fact be a fundamental aspect of obesity and the metabolic syndrome, and

suggest that treatment with a neutral CB1 receptor antagonist will likely improve

Page 20 of 40


the metabolic consequences of obesity in addition to reducing body weight.

Future studies designed to assess the effect of neutral CB1 receptor antagonists

on other parameters of the metabolic syndrome such as cholesterol levels,

insulin resistance, and circulating triglycerides are needed in order to confirm this

hypothesis.

In conclusion, we show that AM4113 is a neutral, high affinity, CB1

receptor antagonist. Our thermoregulation assay demonstrates that this

compound acts centrally to antagonize the effects of a CB1 receptor agonist. The

fact that AM4113 dose-dependently reduces food intake and body weight in rat

supports the view that the endogenous cannabinoid system plays a physiological

role in energy balance. Furthermore, our results suggest that AM4113 may be

substantially less pro-emetic than the CB1 receptor inverse agonist AM251.

Perspectives and Significance

In mammals, several overlapping systems ensure survival by providing the

drive to maintain adequate food intake. These systems converge in the brain,

though many of them lie outside of the CNS, in the gut, liver and adipose tissue.

Endocannabinoids act through central and peripheral CB1 receptors to coordinate

food intake, metabolism and energy expenditure. The lean phenotype of the CB1

receptor deficient mouse (11) supports the idea that this receptor system plays a

central role in these processes. These findings have led to the development of

CB1 receptor compounds for the treatment of obesity. An unresolved issue was

whether activation of the receptor by an inverse agonist contributes to the action

Page 21 of 40


of these compounds. Here we show that a compound with no intrinsic activity

also reduces food intake and body weight in rats. The relative contributions of

central versus peripheral CB1 receptors to these responses remains to be

determined, as does the role of specific neuronal populations possessing central

CB1 receptors. Further development of novel cannabinoid receptor molecules

will allow these questions to be addressed in order to advance our understating

of the physiology of endocannabinoids and their role in food intake and

metabolism.

DISCLOSURE STATEMENT: AM4113 is currently being patent protected by

AM.

ACKNOWLEDGEMENTS

We wish to thank Lorraine Oland for her excellent technical assistance. These

studies were supported by grants from Canadian Institutes of Health Research

(to QJP and KAS) and from National Institutes of Health, U.S.A (DA09158,

DA7215, DA3801 to AM). APC is an Alberta Heritage Foundation for Medical

Research (AHFMR) Graduate Student. QJP and KAS are AHFMR Medical

Scientists. KAS is the Crohn’s and Colitis Foundation of Canada Chair in

Inflammatory Bowel Disease Research and QJP holds a University

Professorship.

Page 22 of 40


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Figure legends

Figure 1. a. Body temperature (mean temp°C ± SEM) in rats pretreated with

either vehicle (n=5), AM251 (5 mg kg-1, n=5), or AM4113 (5 mg kg-1; n=5),

followed by treatment with the CB1 agonist CP55,940 (0.3 mg kg-1) at time 0. b.

The neutral antagonist AM4113 (5 mg kg-1, n=5) and inverse agonist AM251 (5

mg kg-1, n=5) both block hypothermia induced by CP55,940, P<0.05, Newman-

Keuls Multiple Comparison Test.

Figure 2 a. The effect of daily treatment with the neutral antagonist AM4113 (1; 5

mg kg-1, n=5/5) on food intake (mean ± SEM; kcal) in rat. b. Weight gain (mean ±

SEM; g) in rats treated daily with either vehicle or AM4113 (1; 5 mg kg-1), P<0.05,

Newman-Keuls Multiple Comparison Test.* significantly different than vehicle, #

significantly different than 1 mg kg-1.

Figure 3. The effect of AM4113 (10; 20 mg kg-1, n=6/6) on food intake and

weight gain compared with vehicle treated rats (n=6), or, rats treated with the

inverse agonist AM251 (n=6). a. The acute effect of each compound at 1.5, 3, 5,

and 18 h, P<0.05, Newman-Keuls Multiple Comparison Test. b. Daily food intake

(mean ± SEM; kcal) and (c) cumulative weight change (mean ± SEM; g) are

shown before and after the experiment. P<0.05. * significantly different than

vehicle, # significantly different than AM4113 10 mg kg-1.

Page 35 of 40


Figure 4. a. Emesis (mean number of vomiting episodes ± SEM) in ferrets

treated with either vehicle (n=6), AM4113 (10 mg kg-1, n=5), or AM251 (5 mg kg-

1) followed by the emetic M6G (0.5 mg kg-1). The inverse agonist AM251

significantly increased vomiting compared with vehicle treated ferrets, *P<0.05

Newman-Keuls multiple comparisons test. Differences between vehicle treated

ferrets and ferrets treated with the neutral CB1 receptor antagonist, AM4113,

were non-significant. b. The effect of AM4113 (0;5;10;20 mg kg-1) on M6G

induced emesis. Linear regression analysis revealed a positive correlation

between dose and vomiting episodes, P<0.05, however, differences in the

number of vomiting episodes between vehicle and AM4113 treated ferrets was

again not significant.

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A neutral CB1 receptor antagonist reduces weight gain in rat